RU2218571C2 - Reagent for diagnosis of infection caused by virus puumala - Google Patents

Abstract

FIELD: virology. SUBSTANCE: invention relates to diagnosis of virus. Invention proposes reagent for detection of viral infection Puumala using contacting with subject serum infected with virus Puumala and recording agglutination reaction in order to determine the presence of antibodies in serum raised against virus Puumala. Indicated reagent comprises sensitized water-insoluble carrier particles where antigens of virus Puumala are bound with their external surface in the amount from 0.005 to 2 weight %. Reagent is a simple and effective agent for carrying out diagnosis of virus Puumala. EFFECT: improved method for infection diagnosis. 9 cl, 1 tbl, 6 ex

Description

FIELD OF THE INVENTION
This invention relates to a diagnostic reagent for detecting a viral infection. More specifically, it relates to a diagnostic reagent for the serological detection of infection caused by the Puumala virus, which has increased specificity and sensitivity, allowing accurate diagnosis of hemorrhagic fever with renal syndrome caused by the Puumala virus.

Description of the Related Art
Hantaviruses are serologically related members of the Bunyaviridae family. The prototype of the hantavirus is the Hantaan virus, which infects the Asian striped field mouse Apodemus agratius and was discovered in 1976 (Lee et al., "Isolation of the Etiologic Agent of Korean Hemorrhagic Fever," J. Infect. Dis. 137: 298-307 ( 1978)). When a virus is transmitted from mice to humans, an acute illness known as hemorrhagic fever with renal (renal) syndrome (HFRS, Korean hemorrhagic fever) occurs. The Seoul virus and the Puumala virus, discovered later, are also members of the Hantavirus genus. Epidemic hemorrhagic fever found in Asian countries is caused by the Hantaan or Seoul viruses, while HFRS found in European countries is caused by the Puumala virus. The Puumala virus is transmitted through autochthonous European rodent carriers, such as, for example, the bank vole (Clethrionomys glareolus). Recently discovered hantavirus pulmonary syndrome (HPS) in the United States, which is accompanied by high mortality. HPS is caused by the Sin Nombre virus of the same phenotype as the Hantaan virus, but antigenically distinct from it.

 The main clinical features of the epidemic hemorrhagic fever found in Asia and caused by the Hantaan virus or the Seoul virus are fever, hemorrhage (into the digestive tract and subconjunctival) and renal failure syndrome. HFRS, found in Europe, is also characterized by fever, less severe hemorrhage and renal failure syndrome. HPS in the US is also accompanied by fever, acute respiratory distress syndrome, and pulmonary edema, leading to death, approximately one week after the onset of the disease.

 There are large antigenic and genetic differences between natural hantaviruses, so an appropriate vaccine is required for almost every single species. In addition, the high cross-reactivity between individual species of the hantavirus genus makes it difficult to clearly distinguish one hantavirus infection from others when diagnosing.

 Puumala virus infection has been detected in European countries and, in particular, several thousand cases of the disease are reported annually in the countries of Northern Europe. Since its clinical symptoms are less severe than in the case of other hantavirus infections, respiratory distress syndrome and renal failure syndrome make it difficult to distinguish this infection from other cases of respiratory failure or acute nephritis. Therefore, a new, accurate, fast and easy serological diagnostic method for the Puumala virus is needed.

SUMMARY OF THE INVENTION
Therefore, the aim of the present invention is to provide a simple, fast and effective diagnostic reagent for the detection of infections caused by the Puumala virus.

 The present invention provides a diagnostic reagent for detecting infection caused by the Puumala virus, which is a water-insoluble carrier particles and the Puumala virus antigen located on their outer surface.

 The present invention further provides a reagent for detecting infection by the Puumala virus; the reagent is brought into contact with the serum of a patient suspected of being infected with the Puumala virus, and agglutination is observed to determine whether the serum contains antibodies against the Puumala virus, wherein the reagent is a water-insoluble sensitized carrier particles on the outer surface of which are the Puumala virus antigens in amount from 0.005 to 2% weight.

DETAILED DESCRIPTION OF THE INVENTION
For the purposes of this invention, a pure Puumala virus antigen, obtained from tissue culture or animal origin, binds to water-insoluble carrier particles, resulting in sensitized carrier particles.

 Pure Puumala virus antigen can be obtained from various sources, for example, from organs, in particular the brain of animals, or from a tissue culture infected with the virus. In one typical embodiment of the invention, Vero E6 cells are inoculated with Puumala ATCC VR-984 virus and cultured accordingly, and infected cells are lysed. The virus antigen is isolated and purified from the lysate for use in the preparation of the diagnostic reagent of this invention. In another embodiment of the present invention, in which the corresponding animal is used as the source of the antigen of the virus, the Puumala ATCC VR-984 virus is injected into the brain of a newborn hamster (for example, less than one day old), then the infected hamsters are propagated by disintegrating their brain and by isolating and purifying the viral antigen by ultracentrifugation. The viral antigen is used to sensitize water-insoluble carrier particles in order to prepare the diagnostic reagent of this invention.

The virus antigen is exposed to rays and chemically modified to increase its reactivity or sensitivity. For example, by treating about 0.05% (v / v) with a formalin solution at low temperature, for example at 4 ^{° C.} , for several days, for example for 2 weeks, to modify the antigen protein. The antigen thus treated is bound to the outer surface of insoluble carrier particles, for example HDP (high density particles) or red blood cells, to produce sensitized carrier particles. The diagnostic reagent of this invention is an aqueous suspension containing sensitized carrier particles.

Water-insoluble carrier particles may include particles of known inorganic compounds or (red blood cells) animal red blood cells. Particles of inorganic compounds may include, but are not limited to, metal oxides of groups III, IV, or VI of the Periodic Table of the Elements, for example silica, alumina, titanium dioxide, zirconia, sesquioxide iron (F ₂ O ₃ ), iron oxide (F ₃ O ₄ ) cobalt oxide or nickel oxide; hydroxides, for example aluminum hydroxide, iron hydroxide (Fe (OH) ₃ ) or chromium hydroxide; halides, for example silver chloride or silver bromide; sulfides, for example cadmium sulfide; carbonates, for example calcium carbonate or magnesium carbonate, or sulfates, for example barium sulfate or strontium sulfate. Of these, silica, alumina, titania, zirconia or mixed oxides thereof are preferably used.

 Inorganic particulate compounds may also include inorganic oxides, silicon dioxide and oxides of a metal selected from the group consisting of metals of groups II, III and IV of the Periodic System, in which the metal oxides can form a complex with silicon dioxide. Typical examples of inorganic oxides are lithium oxide, sodium oxide, potassium oxide, magnesium oxide, calcium oxide, strontium oxide, barium oxide, aluminum oxide, titanium oxide, zirconium oxide, germanium oxide, hafnium oxide, tin oxide, zinc oxide and the like, but not limited to them.

 Particles of inorganic compounds coated with pigments can also be used for the purposes of this invention, if the pigments do not adversely affect the structure of the inorganic compounds. The compounds may be coated with one or more layers of pigments.

 Particles of inorganic compounds may have an average diameter in the range from 0.1 to 10 microns and preferably from 0.8 to 5.0 microns and a density in the range from 1.5 to 4.0 and preferably from 1.8 to 2.5.

 Preferably, a multilayer coating of particles of inorganic complexes can be used. Complex inorganic particles can be obtained by coating particles of inorganic compounds, for example particles of inorganic compounds based on silicon dioxide, a layer of pigment and, if necessary, an additional layer of the above complex oxides. Then, the resulting particles are treated with binders, for example, silicon and titanium based binders, and a reactive (reactive) surface is formed. Particles thus obtained are commonly referred to as “high density particles” (HDP), and their average diameter is from 0.1 microns to 10.0 microns and the density (specific gravity) is from 1.5 to 4.0.

 For the purposes of this invention, the carrier particles have a dispersibility of 80% or more, and preferably 90% or more. The term "dispersibility" in this context indicates the percentage of the number of individual non-agglomerated particles of the total number of particles suspended in an aqueous medium. Dispersibility can be measured by a conventional meter, such as the Coltercounter Model ZD-1.

 The carrier particles can have a different shape depending on the crystal structure of inorganic compounds and the method of their preparation. Usually they have the shape of a polyhedral, cylindrical, conical and spherical. Preference is given to using particles of a spherical, especially regular spherical shape. These particles can be obtained by known methods described, for example, in JP 52-138094A or JP 61-149644A.

It is possible to use carrier particles impregnated with known dyes. Dyes may include, but are not limited to, cationic dyes such as malachite green, rhodamine B or methylene blue; dispersed dyes such as dianix ^tm (Mitsubishi Chemical Co., Ltd., Japan), disbazole ^tm (ICI, UK, UK) miketonepolyester ^tm (Mitsui Toatsu K.K. Japan); direct dyes, such as Congo red, direct deep black E W or chrysophenin G; Alizarin-Safrol B; acidic dyes, for example, alizarin direct blue A or alizarin-cyarin green G; acidic dyes, for example diamond black F (diamond black F), chromfast navyblue B (chrome navy intensive blue B) or palatine fast blue BN (intense blue); metal-containing dyes, e.g. asidol (azidol) (BASF, Germany), eisen opal (Horoya Kagakusa, Japan) or oreosol (Taoka Kagakusa, Japan); active dyes, for example diamira, michishiron (Mitsubishi Chemical Co., Ltd., Japan) or sumifix (Sumitomo Chemical Co., Ltd., Japan); fluorescent brightening dyes, for example mikiwhite (Mitsubishi Chemical Co., Ltd., Japan) or whitux (Sumitomo Chemical Co., Ltd., Japan). Metal-containing dyes and cationic dyes are preferred, and cationic dyes are most preferred.

 Dyes having a solubility of 1 part by weight or more, more particularly 5 parts by weight, most preferably 10 parts by weight in 100 parts of methanol are used.

 The amount of dyes in the carrier particles may be from 0.5 wt.% To 8 wt.%, And preferably from 1.0 wt.% To 5.0 wt.%, But is not limited to this. Dyes taken in the above interval facilitate the monitoring of antigen-antibody agglutination in order to detect infection by the Puumala virus, and in this case, the outflow of the dye from inorganic carrier particles is also prevented.

 Hemocytes (blood cells) can be used as water-insoluble carrier particles to detect the Puumala virus antibodies of this invention. The source or type of blood cells is not very limited and can be selected from those used to detect antigen-antibody agglutination. Although hemocytes exhibit a relatively lower sensitivity than HDP, they can still be advantageously used as water-insoluble carrier particles for the purposes of this invention.

 Carrier particles with increased sensitivity due to the Puumala virus antibody can be obtained by contacting the virus antigen obtained as described above with carrier particles so as to bind the antigen to the outer surface of the particles. The method of binding the antigen to the particle is not particularly limited, but conventional methods can be used. For example, particles are contacted with an antigen in an aqueous medium, for example, in saline or saline with phosphate buffer (PBS), when the antigen is mixed with suspended particles in an aqueous medium.

The ratio of antigen to carrier particles is in the range from 100 to 400 agglutinating units of HDP and preferably from 150 to 250 agglutinating units of HDP per 1 g of particles. Contacting is carried out at a pH of 6.0-8.0 and at a temperature of from 4 ^{° C.} to 20 ^° C. The term “HDP agglutinating unit” as used herein means the smallest amount of antigen required to detect agglutination of HDP serum.

 After mixing, unbound antigens are removed by washing and the solution is then thermostated with an inert whey protein, such as bovine serum albumin (BSA), to saturate (fill) the remaining active areas of the outer surface of the particles.

 The diagnostic reagent according to this invention can be obtained by suspending sensitized particles in an aqueous solution to a concentration of from 0.005 to 2 wt.% And preferably from 0.05 to 1 wt.%.

 Sensitized carrier particles can be modified to extend shelf life, although the particles themselves are stable, and can be used simply by suspending the modified carrier particles in an appropriate aqueous medium, such as distilled water, immediately before use.

 The diagnostic reagent of this invention is reactive to a serum sample of a patient, preferably infected or susceptible to infection by the Puumala virus, resulting in agglutination with a high degree of specificity and sensitivity.

Examples
The invention will be described in more detail using examples that do not limit the scope of the invention. In particular, water-insoluble carrier particles are prepared by coating a silica core with a mixture of a dye and silica, and then the surface is treated with a silicon binder in one embodiment of the method of the invention, which should not limit the appearance of the water-insoluble carrier particles.

Example 1. Obtaining antigen of the virus Puumala
The Sotkamo strain of the Puumala ATCC VR-984 virus was obtained from the American Cell Culture Collection (MD, USA) and used as a source of the Puumala virus antigen.

 Puumala ATCC VR-984 virus is subcultured in Vero E6 cells (ATCC CRL 1586) for up to 10 passages, and clones that form the largest and most transparent sterile spots are harvested for use as virus blanks. Obtaining a viral antigen is carried out using Vero E6 cells or a newborn hamster less than one day old as host cells. Preferably, a Vero E6 cell is used.

 Obtaining a viral antigen using a Vero E6 cell or a newborn hamster is explained below.

Virus growth in Vero E6 cells
Tissue culture Vero E6 cells were inoculated with 0.1 ml of Puumala virus obtained above and cultured in a thermostat in a CO ₂ atmosphere at 37 ^° C for about 10 days. Infected cells are treated with 0.1% Triton-X and harvested. The collected cells are destroyed by ultrasound and centrifuged at 4 ^o With, receiving a supernatant containing viral particles. The supernatant is treated with gamma rays (3000 rad) to completely inactivate the virus. The inactivated virus is inoculated into Vero E6 cells and assayed by indirect immunofluorescence antibody analysis (IFA) to confirm inactivation of the virus. Ultracentrifugation at a speed of 70,000 rpm was carried out at 4 ^{° C.} for 4 hours to obtain a supernatant containing a viral antigen, which was diluted with PBS and filtered through an ultrafine filter to obtain a purified Puumala virus antigen.

The Puumala virus antigen solution thus obtained is treated with 0.05% (v / v) formalin at 4 ^{° C.} for 2 weeks to modify the surface of the antigen protein in order to increase sensitivity. The antigen is dialyzed against saline and used as an antigenic component of the diagnostic reagent of this invention.

Growing a virus-infected newborn hamster
Puumala virus injection (0.01 ml) is injected into the brain of a newborn hamster (no more than a day old). After 20 days, when more than half of the animals are paralyzed, the animal brain is aseptically removed and homogenized. To obtain an emulsion (10-20% vol.), Physiological saline is added to the homogenate. The emulsion thus obtained from the brain is centrifuged at 600 rpm, 4 ^{° C.} for 30 minutes, and a 10-fold volume of ethanol is added to the supernatant, and then gently shaken at low temperature and dried in vacuum.

 Saline is added to the dried substance of the brain to its initial (brain) volume and 20% (volume) of protamine sulfate. After gentle shaking at low temperature, it is centrifuged at 10,000 rpm for 60 minutes to obtain a supernatant containing a viral antigen free of basic myelin protein. The supernatant was diluted with PBS and filtered through an ultrafine (ultra) filter to give purified Puumala virus antigen.

The Puumala virus antigen solution thus obtained is treated with 0.05% (v / v) formalin at 4 ^{° C.} for 2 weeks to modify the antigen surface protein to increase sensitivity. The antigen is dialyzed against saline and used as an antigenic component for the diagnostic reagent of this invention.

 The titer of the antigen solution thus obtained is determined by ELISA so that the diagnostic reagent containing the antigen can have a constant titer. The titer of the antigen solution obtained in (1) and (2) case is 5-10 agglutinating units of HDP / ml and 120 agglutinating units of HDP / ml, respectively.

Example 2. Obtaining particles with a high density
2800 ml of methanol, 616 ml of an aqueous solution of ammonia (25 wt.%) And 21 ml of an aqueous solution of sodium hydroxide (5M) are poured into a glass flask with a stirrer. At 10 ^{° C.} , 256 ml of a solution (22 wt.%) Of tetraethyl silicate in methanol are added dropwise to the solution at a rate of 25.5 ml / hour, while particles of silicon dioxide with a diameter of 0.91 microns are formed. 625 ml of a solution (44 wt.%) Of tetraethyl silicate in methanol and 625 ml of a solution (1.25 wt.%) Of methylene blue in methanol are simultaneously added dropwise at a rate of 25.5 ml / h, and colored particles of silicon dioxide are obtained. Silica particles are resuspended and decanted to clean and rinse.

The bilayer particles of silicon dioxide / dye thus obtained have an average diameter of about 1.57 microns. The carrier particles are suspended in methanol to a concentration of 0.5 wt.%, And phenyltriethoxysilane is added to the resulting suspension to a concentration of 0.5 wt.%. The reaction is carried out for 16 hours at 10 ^o C and as a result receive particles of silicon dioxide with a modified surface.

Example 3. Obtaining HDP coated with a Puumala virus antigen
To the Puumala virus antigen solution obtained in Example 1, M / 60 and PBS (pH 7.2) were added to dilute the solution to 2 agglutinating units of HDP / ml. The resulting antigen solution (5 ml) was mixed with 0.5% HDP / PBS (5 ml) and sensitized by gentle shaking for 60 minutes. Then, the mixture was washed three times with PBS and suspended by dilution (5 ml) with inactivated rabbit serum in PBS at 56 ^{° C.} for 30 minutes to a concentration of 1% to obtain a sensitized HDP solution.

Example 4. Determination of antibody titer using HDPA or IFA
The serum antibody titer of HFRS patients is determined using HDPA (high density agglutinating particles) as well as indirect IFA (immunofluorescence analysis).

 Each 0.025 ml of dilution from Example 3 is placed in the wells of a V-shaped microplate and 0.025 ml of the test serum is added to the first well (1:10 dilution). The test sample is diluted serially (sequentially), and the sensitized HDP solution (0.025 ml) of Example 3 is added dropwise and mixed well. Leave at room temperature, after 40 minutes, agglutination is observed at each dilution, and the maximum dilution at which antigen-antibody agglutination exists is defined as an “HDP antibody titer”. In this way, an HDP antibody titer is determined for 43 test serum samples.

 In addition, appropriate IFA (indirect immunofluorescence antibody analysis) is performed on 43 test serum samples, and the maximum dilution at which the antigen-antibody fluorescence complex is observed is defined as “IFA antibody titer".

 For comparison, the sensitivity and specificity of IFA and HDPA against Hantavirus infection is determined using the diagnostic reagent disclosed in KR 90-16761A containing HDP sensitized with Hantaan virus.

 The results are shown in the table.

 As can be seen from the table, the results of IFA and HDPA analyzes show that patients in Korea, Japan and China are infected with the Hantaan virus, while patients in Russia, Finland and Yugoslavia are infected with the Puumala virus. IFA and HDPA do not differ markedly in sensitivity and specificity. The diagnostic reagent of this invention enables a simple test to detect the difference between infection with Puumala virus and Hantaan virus with the highest available IFA-appropriate sensitivity. Therefore, it is possible to diagnose the early stage of hemorrhagic fever with renal syndrome (HFRS) caused by the Puumala virus and distinguish it from related syndromes such as acute respiratory distress syndrome or renal failure syndrome, which are difficult to distinguish from HFRS by clinical studies.

Example 5. Obtaining red blood cells sensitized by the Puumala virus antigen
An equivalent volume of tannic acid / PBS 1: 100000 solution was added to PBS containing 2.5% inactivated sheep erythrocytes and the mixture was allowed to react at 20 ^{° C.} for 20 minutes. The reaction mixture is washed with PBS (a mixture of 1/60 M physiological saline with phosphate buffer (pH 7.2) and physiological saline in a volume ratio of 1: 3) once, and then suspended in PBS, a 0.5% suspension containing a tannin-cell complex is obtained blood (hemocyte).

 The tannin-hemocyte suspension was mixed with 80-fold dilution of the Puumala virus antigen obtained in Example 1 (1: 1 by volume), and the resulting mixture was gently shaken at room temperature for 60 minutes to effect sensitization. Then the mixture is washed three times with PBS and suspended in inactivated rabbit serum (1%) in PBS until a concentration of 0.5% is reached, a sensitized erythrocyte solution is obtained.

Example 6. Determination of antibody titer using a sensitized erythrocyte solution
0.025 ml dilution of inactivated rabbit serum was placed in the wells of the V-shaped microplate (Example 3) and 0.025 ml of serum was added to the first well (dilution 1:10; serum No. 11 in the table). The test sample is diluted in batches, a sensitized erythrocyte solution (0.025 ml) is added dropwise in Example 3. After 3 hours at room temperature, agglutination is observed. As a result, the antibody titer is 40.

 Although preferred embodiments of the method of this invention are described above, it should be clearly understood that many variations and / or changes to the basic concepts proposed in the invention, which will be obvious to those skilled in the art, are spiritual and are within the scope of this invention, as reflected in the proposed formula inventions.

Claims (9)

1. A reagent for detecting infection with the Puumala virus, containing sensitized water-insoluble carrier particles and Puumala virus antigens associated with the outer surface of the carrier particles, in an amount of from 0.005 to 2 wt%. 2. The reagent according to claim 1, characterized in that the sensitized water-insoluble carrier particles are suspended in physiological saline with phosphate buffer (PBS) containing 1% (by volume) inactivated rabbit serum. 3. The reagent according to claim 1 or 2, characterized in that said Puumala virus antigens are obtained from Vero E6 cells of a tissue culture or hamster brain. 4. The reagent according to claim 1 or 2, characterized in that said carrier particles are particles of inorganic compounds or red blood cells. 5. The reagent according to claim 4, characterized in that said particles of inorganic compounds are selected from the group consisting of metal oxides of groups III, IV or VI of the Periodic system of elements selected from the group consisting of silicon dioxide, alumina, titanium dioxide, dioxide zirconium, iron sesquioxide (Fe ₂ O ₃ ), iron oxide (Fe ₃ O ₄ ), cobalt oxide and nickel oxide; hydroxides selected from the group consisting of aluminum hydroxide, iron hydroxide (Fe (OH) ₃ ) and chromium hydroxide; halides selected from the group of silver chloride and silver bromide; sulfides; carbonates selected from the group consisting of calcium carbonate and magnesium carbonate; or sulfates selected from the group consisting of barium sulfate and strontium sulfate. 6. The reagent according to claim 4, characterized in that said particles of inorganic compounds are selected from the group consisting of silicon dioxide and metal oxides selected from the group consisting of metals of groups II, III and IV of the Periodic system, said oxides being capable of attaching to silicon dioxide. 7. The reagent according to claim 4, characterized in that said particles of inorganic compounds are high density particles obtained by coating particles of inorganic compounds with a layer of dyes or mixtures of dyes and inorganic compounds, and treated with silicon or titanium binding reagents to obtain a reactive surface, wherein high density particles have an average diameter in the range from 0.1 to 10.0 microns and a specific gravity in the range from 1.5 to 4.0. 8. The reagent according to claim 7, characterized in that said dyes are selected from the group consisting of cationic dyes, disperse dyes, direct dyes, acid dyes, metal-containing dyes and fluorescent brightening dyes. 9. The reagent according to claim 1, characterized in that said antigens are pretreated with formalin at low temperature before binding to particles to increase sensitivity.

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